Molecular motors--kinesin and myosins--play a crucial role in the maintenance and development of the organization, motility, and signaling of healthy cells. Central nervous-system disorders, such as Alzheimer's disease and certain types of cancer, all arise from molecular motors gone awry. There are, however, several fundamental questions about how kinesin-1 moves. (Kinesin-1 is the standard-bearer of kinesins.) Also largely unknown is how Myosin VI moves. To answer these questions we will address the motors at the in vitro, single molecule level. We will apply a number of single molecule tools, including some that have been developed in the first 4 years of this proposal. We will use FIONA (Fluorescence Imaging with One Nanometer Accuracy), DOPI (Defocused Orientation and Position Imaging), and SHREC (Single Molecule High REsolution Colocalization). The theme of the kinesin part is: how is motility affected by each of kinesin's parts, including the head, the coiled-coil, and the tail? The theme of the myosin VI part is: how does such a small motor take such a large step? We will present unpublished data which suggests that kinesin is bound by both heads during a run, making the probability of falling off very low. We will also suggest that the large non-helical region in kinesin-1's coiled-coiled region allows this motor to walk in an asymmetric fashion, allowing the cargo to point forward. Finally, we will suggest that full-length kinesin takes pauses due to the tail-region folding over and interacting with the head region and possibly the microtubule. With regards to myosin VI, we have an enormous amount of preliminary data. It will suggest that the head undergoes a 180 swing during the powerstroke. Furthermore, we suggest that a 3-helix motif in the lever arm uncoils and creates an unprecedented 24 nm extension, which allows the motor to take a 36 nm step. PUBLIC HEALTH RELEVANCE Molecular motors have the job of moving and organizing organelles within a cell. Problems within the motors cause brain cancer, Alzheimer's disease, etc. We seek to understand the basic workings of kinesins and myosin VI, two important motors.